CN103776892B - Matrix with selectivity for micromolecule MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) detection and application thereof - Google Patents
Matrix with selectivity for micromolecule MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) detection and application thereof Download PDFInfo
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- CN103776892B CN103776892B CN201410020300.6A CN201410020300A CN103776892B CN 103776892 B CN103776892 B CN 103776892B CN 201410020300 A CN201410020300 A CN 201410020300A CN 103776892 B CN103776892 B CN 103776892B
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Abstract
The invention discloses a matrix with selectivity for micromolecule MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry) detection and an application thereof. The matrix is graphene oxide grafted 4-vinylbenzeneboronic acid, and takes 4-vinylbenzeneboronic acid as a monomer; the 4-vinylbenzeneboronic acid is grafted to graphene oxide which is subjected to bromine functionalization by triggering atom transfer radical polymerization reaction on the surface, thereby preparing the matrix. The graphene oxide grafted 4-vinylbenzeneboronic acid disclosed by the invention can act as the MALDI-TOF MS matrix for selectively recognizing such micromolecule compounds containing cis-form adjacent dihydroxy structure, as cis-form adjacent dihydroxy compounds in saccharides, phenols and nucleoside; the minimum detection limit can reach 1pmol/mL around, and the selectivity and flexibility are both very high.
Description
Technical field
The invention belongs to the detection technique field of cis o-dihydroxy compound, be specifically related to surperficial Atom Transfer Radical Polymerization synthesis graphene oxide graft copolymerized 4-vinyl phenyl boric acid and detect the application in cis o-dihydroxy compound at MALDI-TOF MS.
Background technology
Grow up the end of the eighties a kind of novel soft ionization biological mass spectrometry and substance assistant laser desorpted/ionization time of flight mass spectrometry (MALDI-TOF MS), and it has, and accuracy is good, sensitivity and resolution high.MALDI-TOF MS is a kind of simple and quick technology, is mainly used in analyzing large biomolecule, and therefore it has been widely used for analysing protein, polypeptide, nucleic acid etc.It makes the pre-treatment step of sample be simplified, and dramatically saves on labour.
In MALDI-TOF MS, matrix plays a part absorption and transfer laser energy, makes sample ionization.But traditional matrix is as easy cracking under laser emission such as CHCA (alpha-cyano-4-hydroxycinnamic acid), DHB (dihydroxy-benzoic acid), thus undesirable matrix ion interference can be produced at low-molecular-weight region (<500Da), make detecting device form saturated focus due to matrix and analysis thing out-phase cocrystallization simultaneously, thus cause the difficulty of micromolecular observation and sign, to such an extent as to be difficult to utilize traditional matrix to go to analyze small-molecule substance.
The physics special based on Graphene and electrochemical properties, it can be used as a class new medium and is successfully captured by analyte, and transfers the energy to analyte by laser emission, in analytic process, analyte can desorb and ionization rapidly, and gets rid of the ion interference of matrix itself well.But also there are some problems using Graphene or graphene oxide as matrix separately.Such as Graphene or graphene oxide can be polymerized in the process of synthesis and application, this severely limits its application.
Summary of the invention
Technical matters to be solved of the present invention is to overcome the shortcoming that traditional MALDI-TOF MS matrix can only analyze macromolecular substances, one is provided to can be used for detecting small-molecule substance, and the selectivity detected and highly sensitive MALDI-TOF MS matrix, simultaneously for this matrix provides a kind of new purposes.
Solving the problems of the technologies described above adopted technical scheme is: this matrix is graphene oxide graft copolymerized 4-vinyl phenyl boric acid, it is prepared by following method: by 4-vinylphenylboronic acid, the graphene oxide ultrasonic disperse of bromine functionality is in 1, in 4-dioxane, under nitrogen protection, add cuprous bromide and pentamethyl divinyl triethylamine, the graphene oxide of bromine functionality and 4-vinylphenylboronic acid, cuprous bromide, the mass ratio of pentamethyl divinyl triethylamine is 1:1 ~ 5:0.2 ~ 0.6:0.2 ~ 0.6, stirring at normal temperature 24 hours, reaction product is through washing, vacuum drying, obtain graphene oxide graft copolymerized 4-vinyl phenyl boric acid.
Mass ratio the best of the graphene oxide of above-mentioned bromine functionality and 4-vinylphenylboronic acid, cuprous bromide, pentamethyl divinyl triethylamine is 1:3:0.44:0.44.
The graphene oxide of above-mentioned bromine functionality can list of references method synthesize, concrete steps are: joined by 0.3g graphene oxide in 15mL anhydrous methylene chloride, ultrasonic 1 hour, then 3mL2-bromine isobutyl acylbromide and 1mL anhydrous pyridine is added, stirring at normal temperature 24 hours, obtains the graphene oxide of bromine functionality.
Above-mentioned have the matrix optionally detected for Small molecular MALDI-TOF MS and contain purposes in the micromolecular compound of cis o-dihydroxy structure at Selective recognition.Its concrete using method is: graphene oxide graft copolymerized 4-vinyl phenyl boric acid is directly used as MALDI-TOF MS matrix and detects the micromolecular compound containing cis o-dihydroxy structure, and the wherein said micromolecular compound containing cis o-dihydroxy structure can be specifically the cis o-dihydroxy compound in sugar, phenol or nucleosides etc.
4-vinylphenylboronic acid is grafted on graphene oxide by the present invention, alleviate the shortcoming of graphene oxide dispersed poor, easy reunion in organic solvent, the graphene oxide graft copolymerized 4-vinyl phenyl boric acid obtained can directly be used as MALDI-TOF MS matrix to the micromolecular compound containing cis o-dihydroxy structure, as the cis o-dihydroxy compound in carbohydrate, phenols, nucleosides etc. carries out high-sensitive selective enumeration method.
Accompanying drawing explanation
Fig. 1 is the mass spectrogram of the graphene oxide graft copolymerized 4-vinyl phenyl boric acid that obtains of embodiment 1 as MALDI-TOF MS matrix.
Fig. 2 is that graphene oxide graft copolymerized 4-vinyl phenyl boric acid detects the mass spectrogram of galactose as MALDI-TOF MS matrix.
Fig. 3 is that graphene oxide graft copolymerized 4-vinyl phenyl boric acid detects the mass spectrogram of wood sugar as MALDI-TOF MS matrix.
Fig. 4 is that graphene oxide graft copolymerized 4-vinyl phenyl boric acid detects the mass spectrogram of adenosine as MALDI-TOF MS matrix.
Fig. 5 is that graphene oxide graft copolymerized 4-vinyl phenyl boric acid detects the mass spectrogram of adenine as MALDI-TOF MS matrix.
Fig. 6 is that graphene oxide graft copolymerized 4-vinyl phenyl boric acid detects the mass spectrogram of catechol as MALDI-TOF MS matrix.
Fig. 7 is that graphene oxide graft copolymerized 4-vinyl phenyl boric acid detects the mass spectrogram of phenol as MALDI-TOF MS matrix.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in more detail, but the invention is not restricted to these embodiments.
Embodiment 1
0.3g4-vinylphenylboronic acid is scattered in 15mL1; in 4-dioxane; then the graphene oxide of 0.1g bromine functionality is added; ultrasonic 10 minutes, then under nitrogen protection, then add 0.044g cuprous bromide and 0.044g pentamethyl divinyl triethylamine; stirring at normal temperature 24 hours; after reacting completely, with methyl alcohol: acetic acid (4:1), deionized water, acetone repeatedly wash to remove the reagent do not reacted completely, last 30 DEG C of vacuum drying.Obtain graphene oxide graft copolymerized 4-vinyl phenyl boric acid.
Prepared graphene oxide graft copolymerized 4-vinyl phenyl boric acid adopts MALDI-TOF MS to characterize, and the results are shown in Figure 1.As seen from the figure, itself is as matrix, little at the assorted peak in low-molecular-weight region, so be very favourable for micromolecular detection.
Embodiment 2
0.1g4-vinylphenylboronic acid is scattered in 15mL1; in 4-dioxane; then the graphene oxide of 0.1g bromine functionality is added; ultrasonic 10 minutes, then under nitrogen protection, then add 0.02g cuprous bromide and 0.02g pentamethyl divinyl triethylamine; stirring at normal temperature 24 hours; after reacting completely, with methyl alcohol: acetic acid (4:1), deionized water, acetone repeatedly wash to remove the reagent do not reacted completely, last 30 DEG C of vacuum drying.Obtain graphene oxide graft copolymerized 4-vinyl phenyl boric acid.
Embodiment 3
0.5g4-vinylphenylboronic acid is scattered in 15mL1; in 4-dioxane; then the graphene oxide of 0.1g bromine functionality is added; ultrasonic 10 minutes, then under nitrogen protection, then add 0.06g cuprous bromide and 0.06g pentamethyl divinyl triethylamine; stirring at normal temperature 24 hours; after reacting completely, with methyl alcohol: acetic acid (4:1), deionized water, acetone repeatedly wash to remove the reagent do not reacted completely, last 30 DEG C of vacuum drying.Obtain graphene oxide graft copolymerized 4-vinyl phenyl boric acid.
Embodiment 4
The graphene oxide graft copolymerized 4-vinyl phenyl boric acid that embodiment 1 obtains is as the purposes of MALDI-TOF MS matrix in Selective recognition galactose.
1mg graphene oxide graft copolymerized 4-vinyl phenyl boric acid is scattered in 1mL ethanol, ultrasonicly makes it be uniformly dispersed, form suspending liquid; By pH value be 8.0 phosphate buffer solution galactose is mixed with the galactose solution of 1mg/mL.Getting 1 μ L suspending liquid drips on MALDI-TOF MS sample target fast, after naturally drying, it drips the galactose solution of 1 μ L1mg/mL again, naturally dries under room temperature condition, carry out MALDI-TOF MS analysis under room temperature condition.Use the same method analysis xylose solution, and analysis result is shown in Fig. 2 and Fig. 3 respectively.As seen from the figure, be that 202.15 places have occurred that galactose adds the molecular ion peak of sodium using graphene oxide graft copolymerized 4-vinyl phenyl boric acid as MALDI-TOF MS matrix at m/z, but can't detect the molecular ion peak of wood sugar, show that graphene oxide graft copolymerized 4-vinyl phenyl boric acid can identify galactose as MALDI-TOF MS matrix, its minimum detectability can reach 0.82pmol/mL.
Embodiment 5
The graphene oxide graft copolymerized 4-vinyl phenyl boric acid that embodiment 1 obtains is as the purposes of MALDI-TOF MS matrix in Selective recognition adenosine.
1mg graphene oxide graft copolymerized 4-vinyl phenyl boric acid is scattered in 1mL ethanol, ultrasonicly makes it be uniformly dispersed, form suspending liquid; By pH value be 8.0 phosphate buffer solution adenosine is mixed with the guanosine solution of 1mg/mL.Getting 1 μ L suspending liquid drips on MALDI-TOF MS sample target fast, naturally after drying under room temperature condition, it drips again the adenosine solution of 1 μ L1mg/mL, naturally dry under room temperature condition, carry out MALDI-TOF MS analysis, use the same method analysis adenine solution, and analysis result is shown in Fig. 3 and Fig. 4 respectively.As seen from the figure, be that 289.26 places have occurred that adenosine adds the molecular ion peak of sodium using graphene oxide graft copolymerized 4-vinyl phenyl boric acid as MALDI-TOF MS matrix at m/z, but can't detect the molecular ion peak of adenine, show that graphene oxide graft copolymerized 4-vinyl phenyl boric acid can identify adenosine as MALDI-TOF MS matrix, its minimum detectability can reach 1.59pmol/mL.
Embodiment 6
The graphene oxide graft copolymerized 4-vinyl phenyl boric acid that embodiment 1 obtains is as the purposes of MALDI-TOF MS matrix in Selective recognition catechol.
1mg graphene oxide graft copolymerized 4-vinyl phenyl boric acid is scattered in 1mL ethanol, ultrasonicly makes it be uniformly dispersed, form suspending liquid; By pH value be 8.0 phosphate buffer solution catechol is mixed with the catechol solution of 1mg/mL.Getting 1 μ L suspending liquid drips on MALDI-TOF MS sample target fast, naturally after drying under room temperature condition, it drips again the catechol solution of 1 μ L1mg/mL, naturally dry under room temperature condition, carry out MALDI-TOF MS analysis, the analysis of Phenol that uses the same method solution, analysis result is shown in Fig. 5 and Fig. 6 respectively.As seen from the figure, using graphene oxide graft copolymerized 4-vinyl phenyl boric acid as MALDI-TOF MS matrix, be that the molecular ion peak of catechol has appearred in 110.11 places at m/z, but can't detect the molecular ion peak of phenol, show that graphene oxide graft copolymerized 4-vinyl phenyl boric acid can identify catechol as MALDI-TOF MS matrix, its minimum detectability can reach 1.34pmol/mL.
Claims (4)
1. one kind has optionally for the matrix of Small molecular MALDI-TOF MS detection, it is characterized in that: described Small molecular is the micromolecular compound containing cis o-dihydroxy structure, described matrix is graphene oxide graft copolymerized 4-vinyl phenyl boric acid, this matrix is prepared by following method: by 4-vinylphenylboronic acid, the graphene oxide ultrasonic disperse of bromine functionality is in 1, in 4-dioxane, under nitrogen protection, add cuprous bromide and pentamethyl divinyl triethylamine, the graphene oxide of bromine functionality and 4-vinylphenylboronic acid, cuprous bromide, the mass ratio of pentamethyl divinyl triethylamine is 1:1 ~ 5:0.2 ~ 0.6:0.2 ~ 0.6, stirring at normal temperature 24 hours, reaction product is through washing, vacuum drying, obtain graphene oxide graft copolymerized 4-vinyl phenyl boric acid.
2. according to claim 1 have optionally for the matrix that Small molecular MALDI-TOF MS detects, and it is characterized in that: the mass ratio of the graphene oxide of described bromine functionality and 4-vinylphenylboronic acid, cuprous bromide, pentamethyl divinyl triethylamine is 1:3:0.44:0.44.
3. according to claim 1 have the matrix optionally detected for Small molecular MALDI-TOF MS and to contain purposes in the micromolecular compound of cis o-dihydroxy structure at Selective recognition.
4. according to claim 3 have the matrix optionally detected for Small molecular MALDI-TOF MS and to contain purposes in the micromolecular compound of cis o-dihydroxy structure at Selective recognition, it is characterized in that: the described micromolecular compound containing cis o-dihydroxy structure is the cis o-dihydroxy compound in sugar, phenol or nucleosides.
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| CN105274000B (en) * | 2014-07-15 | 2017-07-21 | 中国科学院大连化学物理研究所 | A kind of immobilized enzyme reactor and its preparation method and application |
| CN104198572A (en) * | 2014-08-26 | 2014-12-10 | 宜昌捷诺威医药科技有限公司 | Method for detecting content of C18:2CE and SM22:0 lipid composition |
| CN105445399B (en) * | 2014-08-27 | 2017-09-22 | 中国科学院大连化学物理研究所 | A kind of method of the cis o-dihydroxy compound of selective extraction |
| CN106841373B (en) * | 2015-12-07 | 2020-03-10 | 中国科学院大连化学物理研究所 | Application of submicron carbon oxide spheres as matrix in MALDI-MS (matrix-assisted laser desorption/mass spectrometry) |
| CN105954349B (en) * | 2016-06-02 | 2018-11-27 | 南开大学 | A kind of method of qualitative analysis graphene oxide |
| CN109752447B (en) * | 2017-11-01 | 2020-09-01 | 中国科学院化学研究所 | Application of graphdiyne in detection of small molecular substance as MALDI matrix |
| CN111175369B (en) * | 2020-02-24 | 2023-02-03 | 陕西师范大学 | MALDI-TOF MS matrix for small molecule detection and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE102010020933A1 (en) * | 2010-03-10 | 2011-09-15 | Samsung Electro-Mechanics Co., Ltd. | Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-target for the sample analysis, comprises a matrix-assisted laser desorption/ionization time-of-flight plate, a graphene layer, and a metal layer or silicon layer |
| CN102426187A (en) * | 2011-11-21 | 2012-04-25 | 程金生 | Graphene matrix and application of graphene matrix in matrix-assisted laser desorption/ionization-time of flight-mass spectrometry detection |
| CN102603707A (en) * | 2012-02-10 | 2012-07-25 | 中国科学院化学研究所 | 2,3,4,5,-tetra(3',4'-dihydroxyl phenyl)thiophene and application thereof as MALDI (matrix assisted laser desorption ionization) matrix in analyzing small molecules |
| CN103304732A (en) * | 2012-03-14 | 2013-09-18 | 中国科学院大连化学物理研究所 | Monodisperse core-shell structure polymer nano particle as well as preparation and application thereof |
-
2014
- 2014-01-16 CN CN201410020300.6A patent/CN103776892B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE102010020933A1 (en) * | 2010-03-10 | 2011-09-15 | Samsung Electro-Mechanics Co., Ltd. | Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-target for the sample analysis, comprises a matrix-assisted laser desorption/ionization time-of-flight plate, a graphene layer, and a metal layer or silicon layer |
| CN102426187A (en) * | 2011-11-21 | 2012-04-25 | 程金生 | Graphene matrix and application of graphene matrix in matrix-assisted laser desorption/ionization-time of flight-mass spectrometry detection |
| CN102603707A (en) * | 2012-02-10 | 2012-07-25 | 中国科学院化学研究所 | 2,3,4,5,-tetra(3',4'-dihydroxyl phenyl)thiophene and application thereof as MALDI (matrix assisted laser desorption ionization) matrix in analyzing small molecules |
| CN103304732A (en) * | 2012-03-14 | 2013-09-18 | 中国科学院大连化学物理研究所 | Monodisperse core-shell structure polymer nano particle as well as preparation and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Graphene based soft nanoreactors for facile "one-step" glycan enrichment and derivatization for MALDI-TOF-MS analysis;Haihong Bai等;《Talanta》;20130823;第117卷;1-7 * |
| 以石墨烯为基质的MALDI-TOF MS 对有机及药物小分子的检测;程金生等;《分析试验室》;20130531;第32卷(第5期);1-5 * |
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